J Life Sci Res 2018; 16: 9-12
© 2018 Osaka Prefecture University

Journal of Life Science ResearchOnline ISSN 2186-5809

Rapid Communication

Transcriptional regulation of skeletal muscle-specific insulin receptor substrate-1
transcriptHikaru NAGANO † 1, Chinami MATSUYAMA1, Minori SUNAGAWA2, Yuka NAKATSU2, Takeshi NIKAWA3, Shigetada TESHIMA-KONDO1, 2

1

Graduate School of Comprehensive Rehabilitation,school of Comprehensive Rehabilitation, College of Health and Human Sciences,

Osaka Prefecture University, 3-7-30 Habikino, Habikino-City, Osaka 583-8555, Japan2Department of Clinical Nutrition, School of Comprehensive Rehabilitation, College of Health and Human Sciences,

Osaka Prefecture University, 3-7-30 Habikino, Habikino-City, Osaka 583-8555, Japan3

Department of Physiological Nutrition, Institute of Biomedical Science, Tokushima University Graduate School,

Kuramoto-cho 3-18-15, Tokushima 770-8503, Japan
Received 15 October 2018; accepted 13 November 2018
Key words: skeletal muscle; transcriptional regulation; insulin receptor substrate-1 transcript
1 Introduction

line C2C12. We further elucidated the promoter region of theOne of the greatest surprises of high throughput

FL-Irs-1 transcript.

transcriptome analyses is that the mammalian genome ispervasively transcribed into many different complex families

2 Materials and Methodsof RNA. For instance, in protein-coding gene region, there is a

2.1 Cell culture

large number of alternative transcriptional start sites, resulting

C2C12 myoblast cells were cultured in Dulbecco’s Modified

in production of not only protein-coding mRNAs but also long

Eagle Medium (DMEM) containing 10% fetal bovine serum

non-protein-coding RNAs (lncRNA). In addition to lncRNAs,(FBS) and Penicillin-Streptomycin mixed solution (Nacalairecent reports reveal a non-coding function for mRNAs; theTesque, Kyoto, JAPAN) at 37℃ with 5% CO2. At a confluence

untranslated region (UTR) of several mRNA transcripts can alsoof 100%, C2C12 myoblast cells were fused by shifting medium

function as a trans-acting regulatory RNA, which is independent

to DMEM containing 2% horse serum (differentiation medium)

1-3of their protein-coding functions . For instance, we previously

for 3days.

reported that the 5’UTR of Vascular endothelial growth factor

2.2 Plasmid constructs

1(VEGF) mRNA silences Stat1 mRNA expression .

Promoter activity of Irs-1 gene was analyzed using a low

M u s c l e d i ff e r e n t i a t i o n i s a p o w e r f u l s y s t e m f o r

background promoter less Luciferases plasmid pGL3-basic. A 3.6

investigations of lncRNA function and a non-coding function ofkb fragment of the promoter (nt-824 to +2870) was isolated frommRNAs. There is increasing evidence that several lncRNAs andthe mouse Irs-1 promoter (MG2F1)6. pIrs-1 (-824) promoter,the 3’UTR of mRNA regulate myogenesis through modulation

pIrs-1 (-583) promoter and pIrs-1 (-25) promoter were preparedof gene expression required for differentiation, including

by inserting double-strand oligonucleotide Irs-1 prom (-824)

MyoD3. For instance, the 3’UTR of mutant DMPK (dystrophia

Sac1-S, pIrs-1 prom (-583) Sac1-S, Irs-1 prom (-25) Sac1-S andmyotonica protein kinase) mRNA reduces MyoD expression

Irs-1 (-1) -Hind3-AS (Table 1). The PCR products digested withlevels, leading to disruption of C2C12 myogenic differentiation4.

HindIII and SacI restriction enzymes were subcloned upstream

In addition, we previously demonstrated that a transcriptionalof the firefly luciferase gene of pGL3-basic vector (Promega,variant of insulin receptor substrate 1 (Irs-1) acts as a regulatory

Madison, WI) (pGL3-Irs-1 (-824) promoter, pGL3-Irs-1 (-583)

RNA that silences Rb (retinoblastoma) mRNA, resulting inpromoter, pGL3-Irs-1 (-25) promoter).

induction of myogenic dedifferentiation5.

2.3 Quantitative RT-PCR

In the present study, we characterized expression profiles

Total RNA was extracted from C2C12 cells and maleof the two different Irs-1 mRNA (FL-Irs-1 mRNA, Δ5’-Irs-1

C57BL/6 mice (11-weeks-old) from Japan SLC (Shizuoka,mRNA) in several mouse tissues and mouse skeletal muscle cellJapan) isolates tissues (adipose tissue, liver, kidney, heart,skeletal muscle; gastrocnemius, tibialis anterio, extensor†Corresponding author, E-mail: kondoshi@rehab.osakafu-u.ac.jp

digitorum longus) with an acid guanidium thiocyanate-10Table 1phenol-chloroform mixture (ISOGEN; Nippongene, Tokyo,Japan). Quantitative RT-PCR was performed with the primer

Primer sets used in this study

Primer nameand SYBR TM Green dye by using ABI 7300 and 7900 real-SequenceIrs-1 prom (-824) Sac1-S 5’-AAAAGAGCTCGCTAATAGTGCCAGGTGTGAGATCCCAG -3’time PCR system (Applied Biosystems, Foster City, CA), as
described previously5. Standards were run in the same plateIrs-1 prom (-583) Sac1-S 5’-AAAAGAGCTCTACCTGTTGGGTGAGAGCTAGCAGGTC-3’and relative standard curve method was used to calculate theIrs-1 prom (-25) Sac1-S 5’-AAAAGAGCTCGTTGGGGCTCTCGGCAACTCTCCGA-3’

relative mRNA expression. The list of gene-specific primer
sets is shown in Table 1. The Ethics Review Committee forAnimal Experimentation of Tokushima University approved theexperimental protocols described in this study.

Irs-1 (-1)-Hind3-AS

5’-AAAAAAGCTTTCGGAGAGTTGCCGAGAGCCCCAACCA-3’

18S ribosomal RNA-S

5’- GCAATTATTCCCCATGAACG -3’

18S ribosomal RNA-AS 5’- GGGACTTAATCAACGCAAGC -3’

2.4 Transfection
C2C12 cells were transiently transfected with pGL3-Irs-1(-824) promoter, pGL3-Irs-1 (-583) promoter or pGL3-Irs-1 (-25)
promoter using JetPRIMETM transfection regents according to
the manufacturer’s instructions (Polyplus, Berkeley, CA). Cells
were allowed to incubate for 24 hours to experiments.
2.5 Statistical analysis
All data are expressed as the mean ± standard deviation of
3 - 6 individual samples per group. Differences between groups
were analyzed via one-way analysis of variance with SPSS
(release 6.1, SPSS Japan, Tokyo, Japan). Differences between
two groups were tested with Scheffé’s test. P < 0.05 wasFL-Irs-1 mRNA-S

5’- CTATGCCAGCATCAGCTTCC -3’

FL-Irs-1 mRNA-AS

5’- TTGCTGAGGTCATTTAGGTCTTC -3’

Δ5’ Irs-1 mRNA -S

5’- TATGCCAGCATCAGCTTCC -3’

Δ5’ Irs-1 mRNA -AS

5’- TAAAAACGCACCTGCTGTGA -3’

Luciferase-S

5’- TGAGTACTTCGAAATGTCCGTTC -3’

Luciferase-AS

5’- GTATTCAGCCCATATCGTTTCAT -3’

GFP-S

5’- GAAGCGCGATCACATGGT -3’

GFP-AS

5’- CCATGCCGAGAGTGATCC -3’

considered statistically significant.

Figure	  1	A	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  	  B	CDS	3’UTR	intron	  1	Ex	  2	AUG	

5’	CDS	  3’	/	  18	  rRNA	

Ex	  1	

Δ5’	  Irs-­‐1	  mRNA	  3’UTR	

CDS	

5’UTR	GC	TA	EDL	

GC	
TA	
EDL	

Skeletal	  muscle	

Skeletal	  muscle		Δ5’	  Irs-­‐1	  mRNA		

4.0

relative levels	

(fold of control)	

C	

3.0	

2.0

*	

*	

*	FL-­‐Irs-­‐1	  mRNA	

1.5	
1.0	*	 *	 *	

*	
0.5		

1.0	0

Fig. 1

FL-­‐Irs-­‐1	  mRNA	

Δ5’	  Irs-­‐1	  mRNA	TR	

Full-­‐length	  Irs-­‐1	  mRNA	  ＋１�/	  18	  rRNA	

5’UTR	0	1	 2	 3	 1	 2	 3	1	 2	 3	 1	 2	 3	 (days)	

Myoblast	

Myoblast	

Myotube	

Myotube	(A) Schematic representation of two transcriptional variants of mouse Irs-1 mRNAs (FL- and Δ5’Irs-1 mRNAs).
(B) Expression profiles of FL-Irs-1 mRNA and Δ5’Irs-1 mRNA in the indicated mouse tissues. (GC, gastrocnemius; TA, Tibialis anterior;
EDL, Extensor digitorum longus.) Mean ± SD, n=4-6.
(C) Expression levels of FL- Irs-1 mRNA and Δ5’ Irs-1 mRNA in proliferating C2C12 cells (myoblasts) and differentiating C2C12 cells(myoblast and myotubes). Mean ± SD, n=4. *P < 0.05 versus myoblast day 1.

113 Results

its protein are highly expressed (Fig. 1B). In contrast, FL- Irs-1

We first searched transcriptional variants of the mouse

mRNA was specifically overexpressed in several skeletal muscle

Irs-1 gene region using the FANTOM4 database that contains

tissues (gastrocnemius, tibialis anterior, extensor digitorum

mouse transcriptome data. In the mouse Irs-1 gene region, therelongus), compared with other tissues tested (Fig. 1B), suggestingare 19 different transcripts5. Among them, we focused on twothat FL- Irs-1 mRNA may have skeletal muscle-specificdifferent Irs-1 mRNAs (Fig. 1A). One is transcribed from +1

function. In fact, we previously elucidated that FL- Irs-1 mRNA,nt transcription start site, thus it contains full-length 5’UTR

but not Δ5’-Irs-1 mRNA regulates muscle cell differentiation5.

sequence (FL-Irs-1 mRNA). The other is transcribed from +534

We then examined expression profiles of the two Irs-1nt transcription start site, resulting in a short 5’UTR sequencetranscripts in C2C12 cells under proliferation (myoblasts) and(Δ5’-Irs1 mRNA). Both Irs-1 mRNAs contain the same protein

differentiation conditions (myotubes) (Fig. 1C). Expression

cording sequence (CDS) of Irs-1 protein that is fully included inlevels of Δ5’-Irs-1 mRNA were low in proliferation state,

the exon 1.

whereas increased to 3 times during differentiation state (Fig.

We examined expression profiles of the two Irs-1 mRNAs1C). In contrast, expression levels of FL- Irs-1 mRNA werein several mouse tissues (Fig. 1B). Ubiquitous expression of Δ5’-high in proliferation state, but decreased to ~50% duringIrs-1 mRNA was observed in multiple tissues, including skeletal

differentiation state (Fig. 1C), coincident with our previous

muscle, adipose tissue and liver, known that Irs-1 mRNA andobservation that FL- Irs-1 functions under proliferation state5.

Figure 2AFL-Irs1

ATGIrs-1

E box #1∆5’- Irs1

E box #2

LucpGL3-basic vector

LucIrs-1 (-824) promoter*LucIrs-1 (-583) promoter

LucIrs-1 (-25) promoter

0100200300

400500600

Luc/GFP(%)

BIrs-1 (-583) promoter
LucE box #2

E box #1control decoy-ODN
decoy-E-box-#1*

decoy-E-box-#2*0Fig. 250100

150200(%)

Luc/GFP(A) Determination of Irs-1 promoter region in C2C12 cells. Schematic structure of the mouse Irs-1 gene promoter is shown in the top.
Predicted binding sites for E-box are indicated by circles. Schematic representation of luciferase reporter constructs containing
several Irs-1 promoter sequences are shown. Mean ± SD, n=3-4. *P < 0.05 versus pGL3-basic vector.
(B) Effect of inhibition of E-box #1 and #2 sites on promoter activity of FL-Irs-1 mRNA. C2C12 myoblasts were transfected with pIrs1 (-583) reporter construct in combination with the indicated promoter decoy-ODN. Mean ± SD, n=4-5. *P < 0.05 versus control
decoy-ODN.

12We then examined transcriptional regulation of FL-Irs-1

MRF4 and MEF2) activates differentiation-specific genes bymRNA in C2C12 myoblasts. To determine the promoter region

binding E-box elements. In this study, we revealed that thereof FL-Irs-1 mRNA, we used promoter constructs fused towere two regulatory E-box sites in the promoter region of FL-

luciferase reporter gene containing three different 5’-flanking

Irs-1 mRNA. These sites positively regulated FL-Irs-1 mRNA

sequence of the mouse Irs-1 gene, pIrs-1 (-824) promoter, pIrs-transcription (Fig. 2).

1 (-583) promoter and pIrs-1 (-25) promoter (Fig. 2A). The
promoter activity was observed in the pIrs-1 (-583) construct.

5 Conclusions

By contrast, the activity using the pIrs-1 (-824) or the pIrs-1 (-25)

A unique FL-Irs-1 transcript was highly expressed inconstruct was similar to negative control (empty pGL3-basic).

skeletal muscle tissues and cultured myoblasts. In addition,

These results indicated that the promoter regions (-583 to -26)

the transcriptional of FL-Irs-1 mRNA was regulated by E-box-

contained transcriptional activating element, while the promoter

binding transcription factor(s).

region (-824 to -583) contained transcription suppressive
Acknowledgmentselement.
We then searched in silico the canonical binding sites

This work was supported in parts by grants from Grants-(E-box) of muscle-specific transcription factors (MyoD family),in-Aid for Young Scientists (B) (JP17K13071 to HK) from theusing TransSearch program. The activating region (-583 to -26)

Japan Society for the Promotion of Science (JSPS), and The

contained two E-box sites (Fig. 2A). To determine which E-box

Nakatomi Foundation (to HK).

site regulated the promoter activity, we used two E-box decoy
oligonucleotides (decoy-E-box #1, #2) that inhibit binding ofReferencetranscription factors to the E-box site (Table.2). When using

1 Masuda K, Teshima-Kondo S, Mukaijo M, et al. (2008)

pIrs-1 (-583) construct, decoy-E-box #1 and #2 remarkably

A novel tumor-promoting function residing in the 5'

decreased the promoter activity (Fig. 2B). This finding suggests

non-coding region of vascular endothelial growth factor

that E-box #1 and #2 activated FL-Irs-1 mRNA transcription.

mRNA. PLoS Med, 5:e94.
2 Amack JD, Paguio AP, Mahadevan MS. (1999) Cis andtrans effects of the myotonic dystrophy (DM) mutation inTable 2

Oligo nucleotides

NameSequence

E-box #1-S

ACggccagggggtgGA

E-box #1-AS

TCcaccccctggccGT

E-box #2-S

TCcgtcacgtgtttTG

E-box #2-AS

CAaaacacgtgacgGA

a cell culture model. Hum Mol Genet, 8:1975-1984.
3 Amack JD, Reagan SR, Mahadevan MS. (2002) Mutant
DMPK 3'-UTR transcripts disrupt C2C12 myogenic
differentiation by compromising MyoD. J Cell Biol,
159:419-429.
4 Cesana M, Cacchiarelli D, Legnini I, et al. (2011) Along noncoding RNA controls muscle differentiation
by functioning as a competing endogenous RNA. Cell,
147:358-369.
5 Nagano H, Yamagishi N, Tomida C, et al. (2015) A novel

4 Discussion
Previous studies have shown that IRS-1 protein was

myogenic function residing in the 5’ non-coding region

highly expressed in skeletal muscle cell and essential in theirof Insulin receptor substrate-1 (Irs-1) transcript. BMC

5,8

proliferation and differentiation . It has been considered that

Cell Biol, 16:8.

IRS-1 protein is derived from single transcript in skeletal

6 Araki E, Haag BL Matsuda K, et al. (1995)

muscle cells. In this study, we found the existence of a unique

Characterization and regulation of the mouse insulintranscriptional variant of Irs-1mRNA (FL-Irs-1 transcript) thatreceptor substrate gene promoter. Mol Endocrinol.

was overexpressed in skeletal muscle. In addition, FL-Irs-1

9:1367-79.

transcript was mainly expressed in proliferation state of skeletal
muscle cells.
We further elucidated transcriptional regulation of FL-

7 Edmondson DG, Olson EN. (1993) Helix-loop-helix
proteins as regulators of muscle-specific transcription. JBiol Chem, 268:755-758.

Irs-1 transcript in an E-box-dependent manner. Transcription

8 Hakuno F, Yamauchi Y, Kaneko G, et al. (2011)

factors that binds to the E-boxes have been shown to consist

Constitutive expression of insulin receptor substrateof heterodimers between a ubiquitous class A and a tissue-(IRS)-1 inhibits myogenic differentiation throughspecific class B family member of the basic helix-loop-helix

nuclear exclusion of Foxo1 in L6 myoblasts. PLoS One,(bHLH) transcriptional activators2. The muscle-specific class

10:e25655

B members, including MyoD family (MyoD, myogenin,